Exposure of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) at the surface of activated or injured blood cells and endothelium plays a key role in the initiation and regulation of blood coagulation. De novo exposure of these phospholipids (PLs) on injured or apoptotic cells is also implicated in complement activation and in their removal by the RE system. Whereas it is now accepted that sequestration of PS (>PE) to the inner plasma membrane leaflet is mediated by a Mg++ ATPase with specificity for PS (>PE), and that a rise in intracellular Ca++ triggers their rapid migration to the outer leaflet, the detailed mechanisms responsible for this bi-directional translocation of PLs are unknown. Specific Aim 1: To test the hypothesis that egress of PS/PE to the surface of injured or apoptotic cells and the collapse of the normal transbilayer asymmetry is mediated by a Ca++ activated membrane protein (PL scramblase) which promotes rapid PL migration between the outer and inner membrane leaflets. Detergent extracts of RBC membranes will be fractionated and assayed for PL scramblase activity. The protein fraction will be purified, sequenced, and cDNA cloning carried out based on peptide sequence. PL scramblase of platelets and endothelial cells will also be characterized. Specific Aim 2: Analysis of the PL scramblase structure and function. PL scramblase and selected recombinant mutants will be analyzed in terms of Ca++ binding, Ca++ induced conformational change (as determined by CD and differential proteolysis pattern), and the mechanism of transbilayer PL exchange. The role of PIP2 and polyamines on scramblase activity, and the relationship of scramblase to the Scott lymphoblasts will be determined. Specific Aim 3: To identify the protein that functions as the plasma membrane aminophospholipid translocase (APT), which is a Mg++ ATPase. They have developed a mammalian cell line that is suitable for transfection and that is deficient in endogenous APT activity. This APT-deficient cell line will be used to screen candidate cDNAs from human and murine libraries based on their homology to known Mg++ ATPase. Specific Aim 4: Analysis of the APT structure and function, in terms of selectivity for PS (and PE), the putative ATP binding site, phosphorylation site(s), PS-binding site, and its conformational change.